Dendritic cells (DCs) can either sensitize naive T cells via efficiently presenting antigens, or they can tolerize T cells by inducing antigen-specific T cell anergy or regulatory responses. DCs have been largely overlooked as potential therapeutic agents for IDDM as they are not thought to be direct killers of beta-cells. Notably, LPS/anti-CD40 activated DCs express high levels of membrane-associated FasL, which can induce effector T cells to apoptose, and primed pro-inflammatory Th1 cells are particularly sensitive to FasL. Furthermore, activated DCs secrete inhibitory factors, such as TGF-beta, which can down-regulate the functions of T cells and antigen presenting cells. However, immunotherapy based on LPS/anti-CD40 activated DCs lacks antigen specificity, which may cause general immunosuppression. Gene therapy offers a powerful tool to engineer DCs to induce regulatory T cell tolerance to beta-cell antigens. Our preliminary studies have shown that LPS/anti-CD40 activated DCs, which have been transfected with a plasmid encoding a beta-cell antigen, induce antigen-specific Th2 responses in vivo. We hypothesize that administration of LPS/anti-CD40 activated and genetically modified DCs can induce antigen-specific regulatory T cell tolerance, which may inhibit diabetes progression in pre-diabetic NOD mice. In addition, DCs can be directly isolated or generated from bone marrow cells from individual patients, activated and genetically modified ex vivo, and then returned to the patient, making this a very safe clinical procedure. The goals of this proposal are to: 1) determine the optimal conditions for the generation of genetically modified DCs, 2) examine the therapeutic effect of genetically modified DCs on diabetes progression in pre-diabetic NOD mice, 3) determine the mechanism(s) underlying the action of genetically modified DCs in pre-diabetic NOD mice. The results of this proposal will address fundamental questions concerning the interaction of T cells and DCs during an autoimmune response and may provide the basis for novel immunotherapies for the prevention and inhibition of human IDDM.